Abstract Reflecting the highly addictive nature of nicotine, 10-12% of American women smoke during pregnancy. Maternal smoking during pregnancy alters normal lung development to produce lifelong decreases in offspring pulmonary function and increased risk of respiratory diseases. Work from our laboratory and others has shown that almost all the effects of maternal smoking during pregnancy on lung development are mediated by nicotine crossing the placenta to interact with nicotinic receptors in developing lung. This strongly suggests that use of e-cigarettes during pregnancy will have significant detrimental effects on lung development and offspring lung disease; and worse, that nicotine addiction will drive pregnant e-cigarette users to continue use during pregnancy. Further, as well as causing lifelong changes in offspring pulmonary function, in-utero nicotine exposure also appears to alter pulmonary function in subsequent generations, most likely through epigenetic mechanisms. This makes it critically important to characterize the effects of in-utero e-cigarette exposure on offspring lung development and function. Thus, the overall objective of this application is to use a mouse model to characterize the effects of perinatal e-cigarette exposure on offspring pulmonary function and disease and the potential for intergenerational transmittal of the respiratory harms of in-utero e-cigarette exposure. This application represents a collaboration between two outstanding groups, the Pinkerton laboratory which is a world leader in respiratory exposures and the Spindel laboratory, a world leader in the pulmonary effects of in utero nicotine exposure. Our specific aims are as follows: Aim 1. To characterize the direct effect of maternal in-utero e-cigarette exposure on first generation (F1) offspring pulmonary function, respiratory disease and epigenetic changes. This will be done by exposing pregnant BALB/c mice to filtered air, e-cigarettes without nicotine and e-cigarettes with nicotine from gestation day 1 to postnatal day 7 and effects on lung analyzed at 8 weeks of age. In addition, the effects of in-utero exposures on asthma susceptibility will be analyzed using sensitization to house dust mite antigen. Aim 2. To characterize the intergenerational effect of grand-maternal in-utero e-cigarette exposure on second generation (F2) offspring pulmonary function, respiratory disease and epigenetic changes. This will be done as for aim1, but intergenerational effects on the F2 generation will be determined. Aim 3. To characterize the additive, multigenerational effect of both grand-maternal and maternal in-utero e-cigarette exposure on offspring pulmonary function, respiratory disease and epigenetic changes. It is an unfortunate fact that children of smokers are more likely to smoke than children of non- smokers; thus, we will test for the potential of additive harms of multigenerational in-utero e-cigarette exposure. Together these aims will provide a comprehensive analysis of the effects of in-utero e-cigarette exposure on offspring pulmonary function and the potential for multigenerational potentiation of the effects.